Coronaviruses have the largest genomes among known RNA viruses and are phylogenetically divided into four genera. Some betacoronaviruses, such as HKU1, circulate annually in humans and cause mild yet prevalent respiratory disease whereas others, such as SARS-CoV and the recently emerged MERS-CoV, have caused pandemics with high case-fatality rates. Due to their pandemic potential and airborne transmissibility, highly pathogenic coronaviruses are now classified as NIAID Category C priority pathogens. Coronavirus cell tropism and host range are in large part determined by the viral surface spike (S) glycoprotein, which is the largest known class I viral fusion protein. After binding to host receptors and activation by host proteases, the S proteins undergo large conformational rearrangements that result in fusion of the viral and host-cell membranes. A molecular understanding of the structure, function and antigenicity of intact, trimeric S proteins would identify sites of vulnerability that could be targeted by vaccines, therapeutic antibodies and small- molecule antivirals. However, structural studies have been primarily limited to small S protein fragments, which has precluded a unifying structural framework for the biology of coronavirus S proteins. To address this knowledge gap, we have generated soluble, trimeric S proteins from HKU1, SARS-CoV, and MERS-CoV that are amenable to structural analysis by X-ray crystallography and cryo-electron microscopy. We will determine atomic-level structures of these S proteins in the prefusion and post fusion conformations to identify commonalities and differences among divergent betacoronaviruses and define the conformational end-states of the fusion process (Aim 1). With these constructs and a range of biochemical and biophysical assays, we will determine the molecular basis for receptor-induced conformational changes and investigate the effects of host proteases and acidification on this process (Aim 2). The combination of these studies will provide key molecular insights into S protein-mediated membrane fusion and answer long-standing questions regarding S protein triggering. Similar to other class I fusion proteins, such as influenza hemagglutinin (HA) and HIV-1 envelope (Env), coronavirus S proteins are the primary target for neutralizing antibodies and are thus a critical component of developmental vaccines. Currently, the best-characterized antibodies against coronaviruses target the receptor-binding domain (RBD) of the S protein and prevent binding to host cells. The RBD, however, is the most variable part of the spike protein and antibodies that target this domain are unlikely to be cross-reactive, similar to most HA head-binding antibodies. Therefore, we will define the epitopes and mechanisms of antibody-mediated neutralization for novel, non-RBD-directed neutralizing antibodies isolated by our collaborator Dr. Barney Graham (Aim 3). By identifying conserved sites of vulnerability, these studies will provide the foundation for the development of immunotherapies and vaccines that broadly protect against highly pathogenic betacoronaviruses, including those that have yet to emerge.